Extracellular ATP (eATP) is known to act as a danger signal in both plants and animals. In plants, eATP is recognized by the plasma membrane (PM)‐localized receptor P2K1 (LecRK‐I.9). Among the first measurable responses to eATP addition is a rapid rise in cytoplasmic free calcium levels ([Ca2+]cyt), which requires P2K1. However, the specific transporter/channel proteins that mediate this rise in [Ca2+]cytare unknown. Through a forward genetic screen, we identified an Arabidopsis ethylmethanesulfonate (EMS) mutant impaired in the [Ca2+]cytresponse to eATP. Positional cloning revealed that the mutation resided in the
Mitogen-activated protein (MAP) kinase signaling cascades play important roles in eukaryotic defense against various pathogens. Activation of the extracellular ATP (eATP) receptor P2K1 triggers MAP kinase 3 and 6 (MPK3/6) phosphorylation, which leads to an elevated plant defense response. However, the mechanism by which P2K1 activates the MAPK cascade is unclear. In this study, we show that in Arabidopsis thaliana, P2K1 phosphorylates the Raf-like MAP kinase kinase kinase (MAPKKK) INTEGRIN-LINKED KINASE 5 (ILK5) on serine 192 in the presence of eATP. The interaction between P2K1 and ILK5 was confirmed both in vitro and in planta and their interaction was enhanced by ATP treatment. Similar to P2K1 expression, ILK5 expression levels were highly induced by treatment with ATP, flg22, Pseudomonas syringae pv. tomato DC3000, and various abiotic stresses. ILK5 interacts with and phosphorylates the MAP kinase MKK5. Moreover, phosphorylation of MPK3/6 was significantly reduced upon ATP treatment in ilk5 mutant plants, relative to wild-type (WT). The ilk5 mutant plants showed higher susceptibility to P. syringae pathogen infection relative to WT plants. Plants expressing only the mutant ILK5S192A protein, with decreased kinase activity, did not activate the MAPK cascade upon ATP addition. These results suggest that eATP activation of P2K1 results in transphosphorylation of the Raf-like MAPKKK ILK5, which subsequently triggers the MAPK cascade, culminating in activation of MPK3/6 associated with an elevated innate immune response.
more » « less- Award ID(s):
- 2048410
- NSF-PAR ID:
- 10398344
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- The Plant Cell
- ISSN:
- 1040-4651
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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INTRODUCTION Eukaryotes contain a highly conserved signaling pathway that becomes rapidly activated when adenosine triphosphate (ATP) levels decrease, as happens during conditions of nutrient shortage or mitochondrial dysfunction. The adenosine monophosphate (AMP)–activated protein kinase (AMPK) is activated within minutes of energetic stress and phosphorylates a limited number of substrates to biochemically rewire metabolism from an anabolic state to a catabolic state to restore metabolic homeostasis. AMPK also promotes prolonged metabolic adaptation through transcriptional changes, decreasing biosynthetic genes while increasing expression of genes promoting lysosomal and mitochondrial biogenesis. The transcription factor EB (TFEB) is a well-appreciated effector of AMPK-dependent signals, but many of the molecular details of how AMPK controls these processes remain unknown. RATIONALE The requirement of AMPK and its specific downstream targets that control aspects of the transcriptional adaptation of metabolism remain largely undefined. We performed time courses examining gene expression changes after various mitochondrial stresses in wild-type (WT) or AMPK knockout cells. We hypothesized that a previously described interacting protein of AMPK, folliculin-interacting protein 1 (FNIP1), may be involved in how AMPK promotes increases in gene expression after metabolic stress. FNIP1 forms a complex with the protein folliculin (FLCN), together acting as a guanosine triphosphate (GTP)–activating protein (GAP) for RagC. The FNIP1-FLCN complex has emerged as an amino acid sensor to the mechanistic target of rapamycin complex 1 (mTORC1), involved in how amino acids control TFEB activation. We therefore examined whether AMPK may regulate FNIP1 to dominantly control TFEB independently of amino acids. RESULTS AMPK was found to govern expression of a core set of genes after various mitochondrial stresses. Hallmark features of this response were activation of TFEB and increases in the transcription of genes specifying lysosomal and mitochondrial biogenesis. AMPK directly phosphorylated five conserved serine residues in FNIP1, suppressing the function of the FLCN-FNIP1 GAP complex, which resulted in dissociation of RagC and mTOR from the lysosome, promoting nuclear translocation of TFEB even in the presence of amino acids. FNIP1 phosphorylation was required for AMPK to activate TFEB and for subsequent increases in peroxisome proliferation–activated receptor gamma coactivator 1-alpha (PGC1α) and estrogen-related receptor alpha (ERRα) mRNAs. Cells in which the five serines in FNIP1 were mutated to alanine were unable to increase lysosomal and mitochondrial gene expression programs after treatment with mitochondrial poisons or AMPK activators despite the presence and normal regulation of all other substrates of AMPK. By contrast, neither AMPK nor its control of FNIP1 were needed for activation of TFEB after amino acid withdrawal, illustrating the specificity to energy-limited conditions. CONCLUSION Our data establish FNIP1 as the long-sought substrate of AMPK that controls TFEB translocation to the nucleus, defining AMPK phosphorylation of FNIP1 as a singular event required for increased lysosomal and mitochondrial gene expression programs after metabolic stresses. This study also illuminates the larger biological question of how mitochondrial damage triggers a temporal response of repair and replacement of damaged mitochondria: Within early hours, AMPK-FNIP1–activated TFEB induces a wave of lysosome and autophagy genes to promote degradation of damaged mitochondria, and a few hours later, TFEB–up-regulated PGC1⍺ and ERR⍺ promote expression of a second wave of genes specifying mitochondrial biogenesis. These insights open therapeutic avenues for several common diseases associated with mitochondrial dysfunction, ranging from neurodegeneration to type 2 diabetes to cancer. Mitochondrial damage activates AMPK to phosphorylate FNIP1, stimulating TFEB translocation to the nucleus and sequential waves of lysosomal and mitochondrial biogenesis. After mitochondrial damage, activated AMPK phosphorylates FNIP1 (1), causing inhibition of FLCN-FNIP1 GAP activity (2). This leads to accumulation of RagC in its GTP-bound form, causing dissociation of RagC, mTORC1, and TFEB from the lysosome (3). TFEB is therefore not phosphorylated and translocates to the nucleus, inducing transcription of lysosomal or autophagy genes, with parallel increases in NT-PGC1α mRNA (4), which, in concert with ERRα (5), subsequently induces mitochondrial biogenesis (6). CCCP, carbonyl cyanide m-chlorophenylhydrazone; CLEAR, coordinated lysosomal expression and regulation; GDP, guanosine diphosphate; P, phosphorylation. [Figure created using BioRender]more » « less
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SUMMARY Extracellular ATP (eATP) is a key signaling molecule that plays a pivotal role in plant growth and defense responses. The receptor P2K1 is responsible for perceiving eATP and initiating its signaling cascade. However, the signal transduction mechanisms downstream of P2K1 activation remain incompletely understood. We conducted a comprehensive analysis of the P2K1 interactome using co‐immunoprecipitation‐coupled tandem mass spectrometry, leading to the identification of 121 candidate proteins interacting with P2K1. In silico analysis narrowed down the candidates to 47 proteins, including Ca2+‐binding proteins, ion transport‐related proteins, and receptor kinases. To investigate their involvement in eATP signaling, we employed a screening strategy based on changes in gene expression in response to eATP in mutants of the identified interactors. This screening revealed several Ca2+‐dependent protein kinases (CPKs) that significantly affected the expression of eATP‐responsive genes, suggesting their potential roles in eATP signaling. Notably, CPK28 and CPK6 showed physical interactions with P2K1 both in yeast and plant systems. Calcium influx and gene expression studies demonstrated that CPK28 perturbed eATP‐induced Ca2+mobilization and some early transcriptional responses. Overexpression of CPK28 resulted in an antagonistic physiological response to P2K1‐mediated eATP signaling during both plant growth and defense responses to the necrotrophic pathogen
Botrytis cinerea . Our findings highlight CPK28, among other CPKs, as a modulator of P2K1‐mediated eATP signaling, providing valuable insights into the coordination of eATP signaling in plant growth and immunity. -
Abstract The mevalonate pathway plays a critical role in multiple cellular processes in both animals and plants. In plants, the products of this pathway impact growth and development, as well as the response to environmental stress. A forward genetic screen of Arabidopsis thaliana using Ca 2+ -imaging identified mevalonate kinase (MVK) as a critical component of plant purinergic signaling. MVK interacts directly with the plant extracellular ATP (eATP) receptor P2K1 and is phosphorylated by P2K1 in response to eATP. Mutation of P2K1-mediated phosphorylation sites in MVK eliminates the ATP-induced cytoplasmic calcium response, MVK enzymatic activity, and suppresses pathogen defense. The data demonstrate that the plasma membrane associated P2K1 directly impacts plant cellular metabolism by phosphorylation of MVK, a key enzyme in the mevalonate pathway. The results underline the importance of purinergic signaling in plants and the ability of eATP to influence the activity of a key metabolite pathway with global effects on plant metabolism.more » « less
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Abstract Mechanical wounding occurs in plants during biotic or abiotic stresses and is associated with the activation of long-distance signaling pathways that trigger wound responses in systemic tissues. Among the different systemic signals activated by wounding are electric signals, calcium, hydraulic, and reactive oxygen species (ROS) waves. The release of glutamate (Glu) from cells at the wounded tissues was recently proposed to trigger systemic signal transduction pathways via GLU-LIKE RECEPTORs (GLRs). However, the role of another important compound released from cells during wounding (extracellular ATP [eATP]) in triggering systemic responses is not clear. Here, we show in Arabidopsis (Arabidopsis thaliana) that wounding results in the accumulation of nanomolar levels of eATP and that these levels are sufficient to trigger the systemic ROS wave. We further show that the triggering of the ROS wave by eATP during wounding requires the PURINORECEPTOR 2 KINASE (P2K) receptor. Application of eATP to unwounded leaves triggered the ROS wave, and the activation of the ROS wave by wounding or eATP application was suppressed in mutants deficient in P2Ks (e.g. p2k1-3, p2k2, and p2k1-3p2k2). In addition, expression of systemic wound response (SWR) transcripts was suppressed in mutants deficient in P2Ks during wounding. Interestingly, the effect of Glu and eATP application on ROS wave activation was not additive, suggesting that these two compounds function in the same pathway to trigger the ROS wave. Our findings reveal that in addition to sensing Glu via GLRs, eATP sensed by P2Ks plays a key role in the triggering of SWRs in plants.
